Optimization of the extract from flower of Citrus aurantium L. var. amara Engl. and its inhibition of lipid accumulation.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum.

BACKGROUND: Fatty acid synthase (FAS) is a promising antifungal target due to its marked structural differences between fungal and mammalian cells. The aim of this study was to evaluate the antifungal activity of flavonoids described in the scientific literature as FAS inhibitors (quercetin, trans-chalcone, ellagic acid, luteolin, galangin, and genistein) against the dermatophyte Trichophyton rubrum and their effects on fatty acid and ergosterol synthesis. METHODS: The antifungal activity of the natural products was tested by the microdilution assay for determination of the minimum inhibitory concentration (MIC). The effect of the compounds on the cell membrane was evaluated using a protoplast regeneration assay. Ergosterol content was quantified by spectrophotometry. Inhibition of FAS by flavonoids was evaluated by an enzymatic assay to determine IC50 values. Quantitative RT-PCR was used to measure transcription levels of the FAS1 and ERG6 genes involved in fatty acid and ergosterol biosynthesis, respectively, during exposure of T. rubrum to the flavonoids tested. RESULTS: The flavonoids quercetin and trans-chalcone were effective against T. rubrum, with MICs of 125 and 7.5 µg/mL for the wild-type strain (MYA3108) and of 63 and 1.9 µg/mL for the ABC transporter mutant strain (ΔTruMDR2), respectively. The MICs of the fluconazole and cerulenin controls were 63 and 125 µg/mL for the wild-type strain and 30 and 15 µg/mL for the mutant strain, respectively. Quercetin and trans-chalcone also reduced ergosterol content in the two strains, indicating that interference with fatty acid and ergosterol synthesis caused cell membrane disruption. The MIC of quercetin reduced the number of regenerated protoplasts by 30.26% (wild-type strain) and by 91.66% (mutant strain). Half the MIC (0.5 MIC) of quercetin did not reduce the number of regenerated wild-type fungal colonies, but caused a 36.19% reduction in the number of mutant strain protoplasts. In contrast, the MIC and 0.5 MIC of trans-chalcone and cerulenin drastically reduced protoplast regeneration in the two strains. The FAS1 gene was repressed in the presence of MICs of quercetin, trans-chalcone, fluconazole and cerulenin. The ERG6 gene was induced in the presence of MICs of fluconazole and cerulenin and was repressed in the presence of MICs of trans-chalcone and quercetin. Trans-chalcone and quercetin inhibited the enzymatic activity of FAS, with IC50 values of 68.23 and 17.1 µg/mL, respectively. CONCLUSION: Trans-chalcone and quercetin showed antifungal activity against T. rubrum, reducing ergosterol levels and modulating the expression of FAS1 and ERG6.

Effect of dietary supplemental vitamin A concentration on performance, carcass merit, serum metabolites, and lipogenic enzyme activity in yearling beef steers.

A randomized complete block design experiment with 360 single-source black yearling steers (average BW = 316.1 +/- 9.1 kg) fed a 91% concentrate (steam-flaked corn base) diet was conducted to evaluate the effects of supplemental vitamin A (0, 1,103, 2,205, 4,410, or 8,820 IU/kg of dietary DM) on plasma and liver vitamin A and E concentrations, lipogenic enzyme activity, marbling score, and performance of yearling steers. Final BW (586, 580, 590, 585, and 584 kg for 0, 1,103, 2,205, 4,410, and 8,820 IU of supplemental vitamin A/kg of DM, respectively) did not differ (P = 0.39) among treatments. Feed efficiency, ADG, and daily DMI did not differ (P > 0.10) among treatments within each 28-d period or for the overall experiment. From d 57 to slaughter, average DMI (10.33, 10.28, 10.57, 9.75, and 10.22 kg/steer daily for 0, 1,103, 2,205, 4,410, and 8,820 IU of vitamin A/kg of DM, respectively) was less (P < 0.02) by steers receiving 4,410 IU of supplemental vitamin A/kg of dietary DM than for steers in the other treatments. Furthermore, DMI was greater (P = 0.06) for steers receiving 2,205 IU of supplemental vitamin A/kg of dietary DM than for steers receiving 8,820 IU of supplemental vitamin A/kg of DM. Marbling score, HCW, LM area, and 12th-rib fat thickness did not differ (P > 0.10) among treatments. Similarly, the percentage of carcasses grading >or=USDA Choice (62.6, 52.8, 64.0, 58.4, and 58.4% for 0, 1,103, 2,205, 4,410, and 8,820 IU of vitamin A/kg of DM, respectively), Select, or 0.10) among treatments. Except for d 56 (P = 0.050; r = 0.18 for liver retinol), no correlations (P > 0.10) between marbling score and any plasma or liver tissue retinol or alpha-tocopherol concentrations or vitamin A intake were found, and no differences (P > 0.10) in lipogenic enzyme activity were detected among treatments. Taken together with previous and concurrent research, results of this experiment suggest that vitamin A supplementation at a concentration up to twice the NRC recommendation has little effect on performance, marbling, or lipogenic enzyme activity in adipose tissue samples in yearling feedlot steers, and that 2,205 IU of supplemental vitamin A/kg of DM (20,000 IU/steer daily) or less is adequate to meet the vitamin A requirements of finishing beef cattle.

Beneficiary effect of Tinospora cordifolia against high-fructose diet induced abnormalities in carbohydrate and lipid metabolism in Wistar rats.

High intake of dietary fructose has been shown to exert a number of adverse metabolic eff ects in humans and experimental animals. The present study was designed to investigate the eff ect of the aqueous extract of Tinospora cordifolia stem (TCAE) on the adverse eff ects of fructose loading toward carbohydrate and lipid metabolism in rats. Adult male Wistar rats of body weight around 200 g were divided into four groups, two of which were fed with starch diet and the other two with high fructose (66 %) diet. Plant extract of TC (400 mg/kg/day) was administered orally to each group of the starch fed rats and the highfructose fed rats. At the end of 60 days of experimental period, biochemical parameters related to carbohydrate and lipid metabolism were assayed. Hyperglycemia, hyperinsulinemia, hypertriglyceridemia, insulin resistance, and elevated levels of hepatic total lipids, cholesterol, triglycerides, and free fatty acids (p < 0.05) observed in fructose-fed rats were completely prevented with TCAE treatment. Alterations in the activities of enzymes of glucose metabolism (hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and glucose-6-phosphate dehydrogenase) and lipid metabolism (fatty acid synthetase, lipoprotein lipase, and malic enzyme) as observed in the high fructose-fed rats were prevented with TCAE administration. In conclusion, our fi ndings indicate improvement of glucose and lipid metabolism in high-fructose fed rats by treatment with Tinospora cordifolia, and suggest that the plant can be used as an adjuvant for the prevention and/or management of insulin resistance and disorders related to it.

Effects of changes in dietary amino acid balance on milk yield and mammary function in dairy cows.

Two experiments were conducted to determine whether longer-term deficiencies in the supply of limiting amino acids would be accompanied by a decline in mammary function (total DNA, cell proliferation rate and activities of key enzymes), and whether this would adversely affect the cow's ability to respond to a return to a nutritionally adequate diet. The first experiment was performed in early/mid lactation, and the second, using the same cows, was carried out in mid/late lactation. A control group of six cows were given a grass silage-cereal diet containing fish meal as the sole protein supplement (amino acid adequate) throughout the experiments, whereas another group of six cows in treatment received the control diet for 2 wk (lactation wk 5 and 6) and then were changed to a diet in which the fish meal was replaced by an equivalent amount of protein as feather meal (amino acid deficient) for 6 wk before returning to the fish meal diet for 4 wk (Experiment 1). After a rest period of 5 wk, the experimental procedure was repeated (Experiment 2). Although there was a fall in milk yield as lactation advanced, leading to lower milk yields in Experiment 2, the marked difference in milk yield between treatments was similar for the two stages of lactation (21% vs 16% in Experiment 1 and 2, respectively). In both experiments, the marked fall of milk yield in cows given the feather meal diet was completely recovered by a return to the fish meal diet. Despite the markedly lower milk yield with the amino acid-deficient diet, however, there was no clear evidence of corresponding changes in measurements of mammary function.

A new model ELISA, based on two monoclonal antibodies, for quantification of fatty acid synthase.

A new model ELISA, based on two monoclonal antibodies, was developed for the quantification of fatty acid synthase (FAS). In this sandwich assay, a monoclonal antibody M6 was used as a capture on Nunc MaxiSorp ELISA/EIA Modules and another monoclonal antibody M3, labeled with biotin, was used as a detection antibody. More than 10 molecules of biotin were labeled on the anti-FAS monoclonal antibody using modified biotinylation conditions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/mL. Recoveries were 98.54-121.95%, averaging 106.05%. The average FAS concentration obtained from the total 55 healthy volunteers blood was 4.07 +/- 1.81 ng/mL, 4.25 +/- 2.14 ng/mL in women (n = 37) and 3.70 +/- 0.74 ng/mL in men (n = 18). When compared with the previously developed polyclonal-monoclonal ELISA, a different pattern of FAS levels was observed in the supernatant of two cultured breast cancer cell lines in a time course study and there was no linear correlation between the two assays using 215 human blood samples. Thus, this new model FAS-ELISA could be used as an independent assay in measuring clinical samples. In summary, this monoclonal-monoclonal FAS-ELISA is sensitive, accurate, and precise in quantification of fatty acid synthase and has potential as a complementary tool in testing clinical samples.

Modulation of cholesterol levels in broiler meat by dietary garlic and copper.

Male Ross x Ross 208 chickens were fed from hatching to 21 d of age either a control diet (based on corn and soybean meal) or the control diet supplemented with 0, 1.5, 3.0, and 4.5% of a commercial garlic powder in Experiments 1 and 2. Once the dose-response relationship was established, 3% garlic powder or 63 or 180 mg/kg copper as cupric citrate or cupric sulfate pentahydrate were supplemented to the diet (Experiments 3, 4, 5, and 6). In the first two experiments, reductions of plasma cholesterol (P = 0.006) and triacylglycerols (P = 0.013) and liver (P = 0.012) and breast muscle (P = 0.165) cholesterol were observed in garlic-supplemented birds. Feeding either garlic powder or copper (63 and 180 mg/kg) resulted in reduced levels of plasma cholesterol, liver cholesterol, blood reduced glutathione, and breast and thigh muscle cholesterol. Differences were significant at P < 0.05 in at least one experiment. 3-Hydroxy-3-methylglutaryl reductase activity was decreased due to dietary garlic (P = 0.0369), but not by pharmacological levels of dietary copper (P = 0.982). The activity of fatty acid synthetase was decreased in birds fed copper (P = 0.035). Both garlic and copper supplements decreased cholesterol 7 alpha-hydroxylase activity (P = 0.024 and P = 0.022, respectively). The results of these trials confirm the findings that garlic and copper alter lipid and cholesterol metabolism. However, they do not work by the same mechanism. Feeding dietary garlic or copper for 21 d reduced cholesterol levels of broiler meat without altering growth of the chickens or feed efficiency.

Nickel deficiency alters liver lipid metabolism in rats.

The present investigation was designed to examine the effect of nickel deficiency on lipid metabolism in liver and serum lipoproteins of rats. Therefore, a study over two generations was conducted feeding a nickel-deficient diet containing 13 microg/kg nickel or a nickel-adequate diet supplemented with 1 mg/kg nickel. Male 7-wk-old pups from the second offspring were studied. Pups fed a diet poor in nickel tended to have lower weight gains (P < 0.15), nickel concentrations in liver (P < or = 0.1) and iron levels in serum (P < 0.1) than nickel-adequate rats. They were classified as nickel-deficient on the basis of significantly lower erythrocyte counts, hemoglobin concentrations, hematocrits and nickel concentrations in kidney compared with nickel-adequate rats. Nickel deficiency caused a significant triacylglycerol accumulation in liver, with greater concentrations of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids than nickel-adequate rats. Nickel deficiency had slight but significant effects on the fatty acid composition of liver total lipids and phosphatidylcholine and phosphatidylethanolamine. Moreover, nickel-deficient rats had significantly lower activities of the lipogenic enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme and fatty acid synthase than nickel-adequate rats. Nickel-depleted pups had significantly higher concentrations of triacylglycerols and phospholipids in serum VLDL, and cholesterol in serum LDL than nickel-adequate pups. Most of these alterations in lipid metabolism are similar to those obtained in several iron-deficiency studies. Because nickel deficiency also slightly compromised iron status, it is possible that at least some of the observed alterations are due to the moderate iron deficiency.

Dietary medium-chain fatty acids raise and (n-3) polyunsaturated fatty acids lower hepatic triacylglycerol synthesis in rats.

The hypothesis tested was that dietary medium-chain or (n-3) polyunsaturated fatty acids, when compared with (n-6) polyunsaturated fatty acids, alter plasma triacylglycerol levels by affecting hepatic triacylglycerol synthesis as reflected by the activities of acetyl-CoA carboxylase, fatty acid synthase and diacylglycerol acyltransferase in liver. In two separate experiments rats were fed purified diets containing (n-6) polyunsaturated fatty acids in the form of corn oil and either (n-3) polyunsaturated fatty acids in the form of fish oil or medium-chain triacylglycerols (MCT). Consumption of MCT significantly raised plasma triacylglycerol concentrations, whereas fish oil feeding had a lowering effect compared with the corn oil-fed group. In individual rats, the hepatic triacylglycerol concentration was directly correlated with the plasma triacylglycerol concentration (r = 0.60, P < 0.001). The MCT oil diet vs. the corn oil diet markedly raised the activities of hepatic acetyl-CoA carboxylase, fatty acid synthase and diacylglycerol acyltransferase. In the rats fed fish oil, the activities of fatty acid synthase and diacylglycerol acyltransferase were significantly reduced, whereas the activity of acetyl-CoA carboxylase was not affected relative to activities in rats fed corn oil. The activities of the three enzymes were directly correlated with plasma triacylglycerol concentrations in individual rats (r = 0.60-0.75, P < 0.001). The type of fat in the diet probably affects the rate of hepatic triacylglycerol synthesis which is an important determinant of plasma triacylglycerol concentrations.

Polyunsaturated fatty acid-mediated suppression of insulin-dependent gene expression of lipogenic enzymes in rat liver.

The effects of dietary polyunsaturated fat on insulin-dependent gene expression of lipogenic enzymes and a possible mechanism for PUFA-mediated suppression of the gene expression have been investigated in rat livers. When diabetic rats were injected with insulin, the insulin dose-dependent induction of lipogenic enzyme mRNAs were markedly reduced with increasing dietary corn oil. On the other hand, the PUFA-mediated suppression of the mRNA concentrations was partially restored by treatment with pioglitazone, a candidate for increasing insulin receptor phosphorylation. Moreover, insulin binding to receptors of liver, receptor autophosphorylation, and kinase activity toward exogenous substrate were lower in the corn oil diet group than in the hydrogenated fat group. The PUFA-mediated suppression of insulin binding was somewhat restored by pioglitazone, and the suppression of insulin receptor phosphorylation was significantly restored. It is suggested that the PUFA-mediated suppression of insulin-dependent gene expression of lipogenic enzymes can be ascribed to a decrease in insulin receptor binding primarily and also to receptor phosphorylation. Thus, PUFA appears to suppress the lipogenic enzyme gene expression stimulated by insulin.

Hybridization studies of chicken liver fatty acid synthetase. Evidence for the participation in palmitate synthesis of cysteine and phosphopantetheine sulfhydryl groups on adjacent subunits.

Enzymatically inactive variants of chicken liver fatty acid synthetase have been prepared by specific chemical modification of the active cysteine SH group with iodoacetamide, and the phosphopantetheine SH group with chloroacetyl-CoA. Hybridization of each of these variants with the unmodified enzyme yielded (modified)-(unmodified) hybrid dimers which possessed 50% synthetase activity. A 50% active (iodoacetamide-modified)-(chloroacetyl-CoA-modified) hybrid dimer was also demonstrated by recombination of these variants with each other. These results indicate that the two functional sites on the synthetase are independently active, and that each is comprised of a cysteine SH group from one subunit and a complementary phosphopantetheine SH group from the other subunit as depicted by the head-to-tail arrangement proposed by Wakil and co-workers (Wakil, S. J., Stoops, J. K., and Joshi, V.C.